The long-term objectives of this line of research are to: (i) provide evidence in support of adoption of new methods for evaluating water quality and shellfish safety; (ii) develop modification in depuration system that would enhance elimination of bacterial pathogens; (iii) gain a better understanding of the physiological responses of bacteria to suboptimal growth conditions, especially the nonculturable state expressed by Gram- negative bacteria. The specific aims of this specific project are to determine the potential for Escherichia coli and Vibrio vulnificus to enter a viable but nonculturable state under different environmental conditions, to determine the validity of traditional enumeration methods for detecting indicator and pathogenic bacteria in oysters (Crassostrea virginica) by comparing traditional methods with new techniques for direct detection of pathogens, and to evaluate the process of shellfish depuration by monitoring clearance of E. coli and V. vulnificus from oysters. Survival and viability of cultures of the two test pathogens will be evaluated by measuring heterotrophic plate counts, acridine orange direct counts, direct viable counts, fluorescent antibody and fluorescent antibody-direct viable counts, coliform bacteria, electron transport system activity, microcolony formation, and recovery in animals. When appropriate, plasmid profiles can be performed on E. coli (enteropathogneic strain H10407). These methods will be modified and used for detection of the test bacteria in oysters, and the process of oyster depuration will be evaluated Consumption of shellfish constitutes a major cause of food-borne diseases in the U.S. We anticipate better detection of viable pathogenic bacteria in oysters using direct detection methods rather than conventional indicator methods. Better detection of pathogenic bacteria and a better understanding of the ecology and epidemiology of pathogenic bacteria in shellfish are critical for assuring the safety of the shellfish-consuming public.